Background: Ki67, an indicator of proliferation, has been shown to be a useful prognostic and predictive marker for breast cancer. Ki67 can be used to identify two distinct estrogen-receptor positive subtypes: luminal A and luminal B. Luminal A breast cancers have been identified as having a lower proliferation and better outcome compared to luminal B. Furthermore, early clinical trials suggest that Ki67 may be useful in identifying a subset of patients that are sensitive to adjuvant docetaxel treatment. Currently, only estrogen-receptor (ER), progesterone-receptor (PR), and human epidermal growth-factor (HER2) are routinely performed. We have optimized and validated an immunohistochemical (IHC) Ki67 assay and automated computerized image analysis platform for routine clinical testing. Materials and Methods: Immunohistochemical staining was quantitatively assessed using the ACIS® III platform on a cohort (N=761) of tamoxifen treated patients who were diagnosed with breast cancer in Calgary between 1990 and 2001. Tissue microarrays were constructed using three 0.6 mm cores. Ki67 results were available for 510 patients, 461 of which are ER/PR positive and HER2 negative. Staining was performed using the DAKO FLEX ready-to-use system. The percent nuclear area positive was calculated using ACIS III and the maximum value was used in statistical analysis Results: X-tile statistical software was used to identify an optimal Ki67 cut point to distinguish differential overall survival in node negative ER positive cancers of 18.75%. This cut point was then used to categorize the 461 ER/PR positive and HER2 negative breast cancers into luminal A (407; 88.3%) or luminal B (54; 11.7%) subtypes. The 8-year breast cancer specific survival was 85.2% (95% CI = 81.3% - 89.1%) for luminal A and 53.6% (95% CI = 39.3% - 67.9%) for luminal B (P<0.0001). Cox regression showed a hazard ratio of 1.63 (95% CI = 0.99 — 2.67, p=0.055), adjusting for age, tumor size, grade and lymph node status. Discussion: Quantification of Ki67 expression using automated image analysis can be used clinically to distinguish luminal A from luminal B in ER/PR positive and HER2 negative breast cancers. The purpose of this project was to develop a reliable Ki67 assay that can be easily adopted by other testing centers. Using a ready-to-use IHC system — such as DAKO FLEX — allows for consistent results between other clinical laboratories. Additionally, using an automated, quantitative imaging system — such as the ACIS® III — reduces inter-observer variation that can occur by human visual assessment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-35.

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Background: It has been repeatedly demonstrated that ER-positive patients have a significantly increased response to tamoxifen, however, continuous association between relative risk and quantitative ER expression has yet to be fully established. Additionally, although it has been hypothesized that estrogen receptor positivity and tamoxifen treatment may play a role in radiation therapeutic response, an assessment of whether quantitative levels of ER predict response to radiation therapy has yet to be determined. Materials and Methods: Fluorescence immunohistochemistry with AQUA® technology was used to quantitatively assess ER expression on a cohort (n = 568) of retrospectively collected breast cancer specimens from patients treated with tamoxifen (20mg) ± radiotherapy (RT). Staining, image acquisition and AQUA analysis was performed at two independent sites using two different standardized digital pathology platforms. AQUA technology is a completely standardized and objective platform with minimized operator interaction that provides tumor-specific, quantitative and continuous expression score data. Results: A comparison between completely independent sample staining and quantitative assessment at two sites showed highly significant correlation, with AQUA score values approaching unity (Pearson's R=0.94; linear slope = 0.999) and indistinguishable means (p=0.93). Assessment of the same tissue slides on two different instrument platforms (HistoRx PM2000 v. Aperio Scanscope FL) also showed highly significant correlation (Pearson's R=0.95; linear slope = 1.01; p = 0.89). Continuous ER AQUA scores showed a highly significant association with 5-year disease-free survival by itself (HR = 0.80 (95%CI: 0.70-0.91); p=0.001) and when put into a model with nodal status and tumor size (HR=0.80 (95%CI: 0.68 — 0.94); p=0.006). The cohort was then divided at the median AQUA score representing relative low and high ER expressing patients. The low ER expressing group showed significant benefit from RT for 5-year disease-free survival (HR = 0.56 (95%CI: 0.32-0.95); p=0.03) and maintained significance at the 10% level when nodal status and tumor size were adjusted for in the model (HR = 0.60 (95%CI: 0.32 — 1.10); p=0.097). In contrast, the high ER expressing group showed no benefit (HR = 0.81 (95%CI: 0.43-1.52); p=0.51) for radiation treatment. No benefit for either group was observed for 15-year overall survival. Discussion: Taken together, these data demonstrate that quantification of ER, beyond simple positive/negative characterization, could provide valuable predictive information for the treatment of breast cancer, specifically for predicting a group more likely to respond to radiation therapy and sparing patients from a potentially harmful treatment. These data will need further validation on an independent cohort designed to differentiate radiation response. Furthermore, these data indicate that true quantification of ER expression provides a continuous recurrence risk assessment for patients being treated with tamoxifen. Because these data are standardized across sites and imaging platforms, misclassification of patients is significantly reduced as compared to the current standard by which ER expression is determined. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD10-08.

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